TOXICITIES OF CALCINEURIN ANTAGONISTS

Chronic Cyclosporine Nephrotoxicity: A Pig Model

D. Cibulskyte, H. Kaalund, M. Pedersen, A. Hørlyck, N. Marcussen, H.E. Hansen, M. Madsen,
and J. Mortensen

ABSTRACT
Cyclosporine A (CsA) is one of the keystones in immunosuppressive treatment after solid
organ transplantation, despite its major side effects such as nephrotoxicity. The chronic
nephrotoxic effects of CsA seen in humans have been difficult to reproduce in small-animal
models. The aim of the present study was to examine the chronic nephrotoxicity produced
by therapeutic dosages of CsA in a pig model. Among 11 Gottingen minipigs included in
the study, three died, yielding data from five animals given CsA (10 mg/kg/d, orally) for 6
months, and three controls. Body weight, blood pressure, glomerular filtration rate (GFR)
by plasma clearance of 51Cr-ethylenediamine-tetraacetic acid, CsA concentration, serum
creatinine, and other values were measured every 5 weeks. Our results showed that the
whole blood trough CsA levels were lower in pigs than in humans treated with similar CsA
doses. Renal biopsies, which were obtained successfully, except one case of macroscopic
hematuria, showed no histological changes in the kidney. No significant increase in serum
creatinine or blood pressure was observed. Surprisingly, there was a significant increase in
GFR during CsA treatment. We conclude that the pig model displays a hyperfiltration that
warrants further investigation.

C YCLOSPORINE A (CsA) is an important immuno-

suppressive drug used in organ transplantation as well
as in the treatment of some primary renal and nonrenal
immune-mediated diseases. CsA has led to a significant
reduction in renal allograft rejection, improving graft and
patient survival after its introduction for general use in the
early 1980s.1–3 However, the drug has some serious side
effects, particularly chronic nephrotoxicity.4 –9 This condi-
tion is manifested by renal insufficiency due to glomerular
and vascular changes, abnormalities of tubular function as
well as hypertension. Renal biopsy reveals arteriolar hyli-
nosis, focal interstial fibrosis, tubular atrophy, and glomer-
ulosclerosis with focal atrophy.
One of the reasons for the limited success of experimen-
tal research studying the mechanism of chronic CsA neph-
rotoxicity is the lack of a suitable animal model. CsA side
effects have been observed in a number of species, including
goat, sheep, baboon, dog, guinea pig, rabbit, and rat.10 The
From the Departments of Renal Medicine (D.C., H.E.H., M.M.),
Magnetic Resonance (M.P.), and Radiology (A.H.), Skejby Syge-
hus, Aarhus University Hospital, Aarhus, Denmark; Department
of Clinical Biochemistry (H.K.), Odense University Hospital,
Odense, Denmark; Department of Pathology (N.M.), Aarhus
Sygehus, Aarhus University Hospital, Aarhus, Denmark; and
Institute of Experimental Clinical Research (J.M.), Skejby Syge-
hus, Aarhus University Hospital, Aarhus, Denmark.
Address reprint requests to D. Cibulskyte, Department of
Renal Medicine, Aarhus University Hospital, Skejby Sygehus,
8200 Aarhus N, Denmark. E-mail: donataci@yahoo.com

0041-1345/05/$–see front matter © 2005 by Elsevier Inc. All rights reserved.

doi:10.1016/j.transproceed.2005.09.004 360 Park Avenue South, New York, NY 10010-1710

3298 Transplantation Proceedings, 37, 3298 –3301 (2005)

CHRONIC CYCLOSPORINE NEPHROTOXICITY
majority of studies using rodents as experimental models
have failed to show chronic CsA nephrotoxicity as seen in
humans treated with therapeutic doses.11 The structural
damage was only induced at CsA12–13 high doses or in
salt-depleted rats, which have increased susceptibility to the
effects of CsA.14 In contrast, a study in Sprague-Dawley rats
given 10 mg/kg of CsA intraperitoneally daily for 2 weeks
showed renal tubular dilatation, suggesting subtle tubular
damage and a precursor of focal interstitial fibrosis.15 The
renal functional and histological changes have also been
produced in three other studies, where Sprague-Dawley
rats were administered 5 mg/kg/d, 12.5 mg/kg/d, or 15
mg/kg/d orally.16 –18
Experimental research on CsA nephrotoxicity has also
been performed in rabbit models, but far less frequently
than in the rat.11 One study in New Zealand rabbits used
low doses of CsA (2.5, 5, or 10 mg/kg/d, intravenously),
revealing morphological changes similar to those seen in
man.19 The pig has been used to elucidate the mechanisms
of the functional nephrotoxic effects of CsA.10 Vaden et al
produced mild multifocal renal tubular dilatation in York-
shire pigs, which received 25 mg/kg/d CsA orally for 22
days.20
The pig has several advantages as a model for pharma-
cological studies,21 because of similar cardiovascular and
renal physiology and anatomy to man.22 From an economic
point of view, it is obvious that the minipig is attractive
because of its low weight at birth, sexual maturity, and adult
age. Ethically, such experiments cannot be performed in
man. However, there are some differences in the pharma-
cokinetics of immunosuppressive drugs between pigs and
humans. Measuring CsA and prednisolone levels by high-
pressure liquid chromatography after both intravenous and
oral doses, Frey et al23 reported that to obtain the same
target CsA concentrations, pigs required two and four to six
times higher doses, respectively. The aim of this work was to
elucidate the nephrotoxic effects after 6 months of the CsA
dosage used after clinical kidney transplantation.
MATERIALS AND METHODS
Eleven Gottingen minipigs of body weight of 20 kg were housed in
individual cages on a farm and fed a standard pig diet. After the
baseline evaluation, six animals were administered CsA (Sandim-
mun Neoral oral solution, Novartis Pharmaceuticals, Basel, Swit-
zerland; 10 mg/kg/d) in two divided oral doses. From the evening
before the investigations, the pigs abstained from food but had free
access to tap water.
The animals were premedicated with azaperon (1 mg/10 kg) and
midazolam (1 mg/kg) intramuscularly. Anesthesia was induced by
etomidate (0.25 mg/kg intravenously) and maintained with keta-
mine (6 mg/kg/h intravenously) and isofluran (0.5% to 0.75% in a
volumetric mixture of O2 and N2O in a ratio of 1:1). The pigs were
orotracheally intubated and mechanically ventilated with a respi-
rator (Siemens, Servo 900D). Through an ear vein isotonic saline
alternating with 5% glucose (10 mL/kg/h) was given to maintain
hydration. Ampicillin (1 g intravenous) was delivered at both the
beginning and the end of the investigations. For urine collection
and measurement, a 12-F catheter was placed transurethrally into
3299
the bladder. Through a small incision the superficial femoral artery
was cannulated for blood sampling and measurement of blood
pressure. At baseline (T0) and at 5-week intervals (T1 to T5), a
battery of examinations was performed over 6 months.
The animals were weighed upon arrival. Blood samples were
collected 18 hours after latest CsA dose for determination of serum
creatinine and of whole blood trough CsA concentrations using the
EMIT 2000 Cyclosporin kit (Dade Bering). Blood pressure was
measured using a pressure transducer (Statham Gould #4523551)
attached to a monitor (Medistim CardioMed CM-4008, Norway).
Glomerular filtration rate (GFR) was calculated by plasma clear-
ance of 51Cr-ethylenediamine-tetraacetic acid (EDTA) (Behring,
Marburg, Germany). A bolus injection of 6 MBq 51Cr-EDTA was
given intravenously followed by continuous infusion of 1.5 MBq
51
Cr-EDTA/h during the experiment. After a loading period of 1
hour, a clearance period of 1 hour was used. The bladder was
emptied at the beginning and at the end of the collection period.
Plasma 51Cr-EDTA was obtained at time 0 as well as at 20, 40, and
60 minutes. The values of plasma and urine 51Cr-EDTA were
estimated in a gamma counter (Cobra, Packard, USA) and cor-
rected for radioactive decay. GFR was calculated as:
GFR ⫽ UCr ⫻ V ⁄ PCr
where UCr is the concentration of 51Cr-EDTA in the collected
urine, V is the urine flow per unit of time, and PCr is the
interpolated plasma 51Cr-EDTA concentration of the four blood
samples.
Finally ultrasonography was performed to measure renal length
and gross morphology in addition to taking two kidney biopsies
from the lower pole using a Bard 18-G cannula. The frequency of
complications as urinary infection, bleeding and infection after
incision over the femoral artery and after renal biopsies was
recorded. Upon completion the animals were awakened and taken
back to the farm.
At the final experiment a bilateral nephrectomy was performed.
The animals were sacrificed using a lethal dose of potassium
chloride. Kidney tissue and biopsies were examined for the follow-
ing histopathological changes: (1) arteriolar hyalinosis, (2) patchy
interstitial fibrosis, (3) glomerulosclerosis, and (4) tubular atrophy.
The study protocol was approved by the Animal Ethics Council.
We used one-way repeated measures analysis of variance
(ANOVA) and Wilcoxon signed-rank test to assess changes within
the CsA-treated group. Comparisons between two groups were
performed using the Mann-Whitney test. A significance level of P
⬍ .05 was chosen.
RESULTS
Eleven Gottingen minipigs were included in the study: six
were given CsA and five were used as untreated controls.
Three animals died: one CsA-treated pig experienced re-
spiratory insufficiency due to a technical problem during
anesthesia and two controls developed fulminate pulmo-
nary infections. Data were therefore obtained from five
animals in the CsA group and three in the control group.
In all pigs two biopsies of sufficient size were obtained
without complication, except one case of macroscopic he-
maturia for 2 hours. The femoral incision healed in all cases
without complications.
The results of our study are shown in Table 1. From T0
to T1 the CsA-treated pigs lost weight on average from
20.5 to 18.3 kg (P ⬍ .03). Thereafter they gained weight
3300 CIBULSKYTE, KAALUND, PEDERSEN ET AL

Table 1. Functional and Chemical Parameters (Mean ⴞ SD) for the CsA-Treated Pigs Measured Baseline (T0) and Every
5 Weeks (T1–5) for 6 Months
Time Body weight (kg) GFR (mL/min) CsA (ng/mL) Creatinine (␮mol/L) Mean blood pressure (mm Hg)

T0 (0 wk) 20.2 ⫾ 1.3 48 ⫾ 10 90 ⫾ 10 79 ⫾ 18

T1 (5 wk) 17.8 ⫾ 1.4 44 ⫾ 15 81 ⫾ 35 68 ⫾ 10 75 ⫾ 18
T2 (10 wk) 18.1 ⫾ 2.1 87 ⫾ 25 27 ⫾ 8 65 ⫾ 16 77 ⫾ 12
T3 (15 wk) 19.8 ⫾ 2.6 113 ⫾ 76 49 ⫾ 24 94 ⫾ 29 81 ⫾ 14
T4 (20 wk) 21.6 ⫾ 2.9 67 ⫾ 16 53 ⫾ 40 83 ⫾ 8 82 ⫾ 8
T5 (25 wk) 23.1 ⫾ 3.1 100 ⫾ 27 64 ⫾ 57 78 ⫾ 15 80 ⫾ 10

for the rest of the experiment to an average weight of
23.0 kg. The increase from T1 to T5 was significant (P ⬍
.05). The changes in weight from T1 to T5 in the
CsA-treated group were significantly different from the
changes in the control group (P ⬍ .05), but there was also
a significant difference in weight between the two groups at
T0 (P ⬍ .05).
Serum creatinine did not increase and mean blood
pressure was constant during the study (P ⫽ .940). GFR
increased significantly during CsA administration (P ⫽
.031). The values were the same at T0 and T1 with a mean
value of 46 mL/min. Subsequently, GFR increased steadily
for the rest of the experiment, reaching a value of 100
mL/min at T5.
Due to an insufficient number of 51Cr-EDTA clearance
results in the control group these data are omitted; we refer
to the corresponding GFR estimates based on magnetic
resonance imaging (MRI) GFR data (Fig 1).24 The average
CsA whole blood trough concentration was 81 ng/mL at 5
weeks and then varied around 50 ng/mL for the rest of the
study period (Table 1). However, there were no significant
differences (P ⫽ .05). The kidney specimens showed no
Fig 1. The relative GFR values for the CsA-treated group (grey
columns) and the control group (black columns) measured at
baseline (T0), 15 weeks (T3), 20 weeks (T4), and 25 weeks (T5).
vessel changes, no patchy interstitial fibrosis, no glomeru-
losclerosis, and no tubular atrophy.
DISCUSSION
As an experimental animal for the study of CsA-induced
nephrotoxicity, the pig has advantages over the rat because
of its similarity to humans in cardiovascular as well as renal
physiology and anatomy. Furthermore, rat studies do not
permit serial biopsies. Previously, the rat has been popular
as an animal model because it is small, cheap, and has a fast
turnover, so that studies can be performed quickly, which is
one of the drawbacks of the pig. The Gottingen minipig,
however, has an acceptable size for scientific investigations.
A 10 mg/kg/d dosage of CsA is often used in clinical
practice after renal transplantation. We chose this dosage
for the experiment to observe effects during 6 months of
treatment of CsA in an animal model reflecting humans.
Whole blood trough concentrations of CsA were lower than
in humans, due to differences in drug pharmacokinetics.
The animals did not develop diarrhea. Little is known about
the dose of CsA, which can induce the nephrotoxic effects in
the pig. In one study, pigs that received 25 mg/kg/d CsA
orally developed renal tubular dilatation, with blood 24-
hour trough concentrations measured by radio immunoas-
say being maintained at 641 ⫾ 175 ng/mL.20 Frey et al
found that the pig shows a large variability in the bioavail-
ability of CsA delivered by the oral route.23 The pig has also
a clearance rate that is considerably higher than that in
man. These findings may be attributable to different rates of
renal or biliary clearance, species-specific metabolic path-
ways, or differences in analytical methods.20
Surprisingly, the present study showed glomerular hyper-
filtration in the CsA-treated group starting between week 5
and 10. To our knowledge, this has not been described
previously during long-term CsA treatment; at present it is
unknown whether the observation is caused by CsA. In
diabetic nephropathy, renal function impairment is pre-
ceded by renal growth24 followed by hyperfiltration25 and
ultimately ending with deteriorating kidney function lead-
ing to end-stage renal failure. It is tempting to speculate
that the observed hyperfiltration found in this study may be
induced by CsA and might indicate an early phase of CsA
nephrotoxicity. Further studies to evaluate these issues and
the pharmacokinetics of CsA in the pig are in progress.
To conclude, our work indicates that the pig may be
CHRONIC CYCLOSPORINE NEPHROTOXICITY
suitable as an animal model for studying the nephrotoxicity
of CsA. The increase in GFR during CsA treatment war-
rants further investigation.
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